Leukocyte recruitment is central to inflammation, and there is intense focus on inhibiting the process both for chronic inflammatory disorders and for conditions where inflammation plays a contributory role, such as atherosclerosis. Whereas current anti-inflammatory drugs such as NSAIDs target secondary effects that are induced after inflammation is already established, inhibiting leukocyte recruitment would prevent the initiation of inappropriate inflammatory responses. Chemotaxis - the movement of cells aligned with a chemical gradient - is the fundamental process underlying neutrophil recruitment. Despite its importance, there are limited screening tools available for medium to high throughput analysis of leukocyte recruitment. Typically, primary screening incorporates a molecular target such as an enzyme or receptor, and hits are then validated in cumbersome in vitro cell migration assays. Existing assays suffer from three main problems: 1) Lack of repeatability (e.g. due to trapped air), 2) need for manual operations (e.g. filter removal), and 3) large cell number requirement (e.g. 1x105 cells/datum). We aim to solve each of these problems by taking advantage of microfluidic technology. In this phase II project we will establish injection molding of full plate (i.e. 96 unit) parts and adapt manufacturing methods developed under the phase I grant to produce devices that suitable for automation. We will use these devices to show reproducibility and data quality while reducing the number of cells required per datum 10-fold. Importantly we will demonstrate the use of the microfluidic chemotaxis device in a high content screening study where multi-parametric image- based analysis is applied to test both migration and polarization of primary human neutrophils. PUBLIC HEALTH RELEVANCE: The migration of cells from the bloodstream to sites of injury is one of the fundamental processes underlying chronic inflammatory disorders such as arthritis and inflammatory bowel disease. Responding cells migrate toward higher concentrations of signaling molecules that are released at the injury site. To enable the discovery of drugs to block this process, we are developing plastic devices for high throughput cell migration assays in defined chemical gradients.